Vectors based on adeno-associated viruses (AAV) have proven to be a convenient tool for genomic editing. However, for transgenesis of laboratory animals or gene therapy for human diseases, a high AAV titer is required. We optimized the conditions for producing AAV serotype 9 (AAV9) to increase the yield of the virus. A common approach is based on transfection of producer cells with three plasmids. We performed transfection with two plasmids encoding all the necessary components for AAV9 production. This allowed us to increase the final title fourfold. Optimization of the molar ratio of the plasmid containing the gene of interest to the plasmid encoding the proteins of the replicative complex, capsid, and auxiliary factors led to a twofold increase in the titer. Optimization of the composition of the cultivation medium for AAV9 production allowed us to increase significantly the yield of the virus. When the DMEM-F12 production medium with a less nutritious DMEM was supplemented with fetal bovine serum, the yield of AAV9 increased by approximately three orders of magnitude. Thus, optimization of the number and ratio of plasmids, as well as the composition of the cell culture medium, made it possible to increase significantly the production of AAV9 and achieve a final titer of 2.5 x 1012 vector genomes in 1 mL of the production medium of the adhesive HEK293T cell line.